Corneal endothelial cell dysfunction causes decreased vision and can only be treated by corneal transplantation to replace human corneal endothelial cells (HCECs), which do not proliferate in vivo. Over 15,000 corneal transplants are performed every year in the United States for endothelial cell dysfunction, but with increasing tissue demand and threats to the tissue supply, a donor tissue crisis is imminent. To meet future demand, the donor pool can be expanded by transplanting cultured endothelial cells, but novel methods of delivering cultured cells to the eye are needed for cell transplantation to be successful. The goal of our research program is to develop a method of HCEC transplantation to posterior corneal stroma without transferring cells on a membranous cell carrier. We hypothesize that transplantation of HCECs to posterior corneal stroma will be facilitated if cells are drawn into direct contact with the stroma. In preliminary studies, we have successfully incorporated superparamagnetic microspheres (SPMs) into cultured HCECs in vitro and demonstrated cell migration toward a magnetic source. We will localize HCECs with incorporated SPMs to the corneal stroma by applying an external magnetic field, eliminating the need for a membranous cell carrier. The major benefit of this approach will be the much needed expansion of the donor pool, but in addition, the proposed technique will be minimally invasive and sutureless when applied to humans in vivo, and promoting cell attachment directly to corneal stroma will enable treatment of diseases, such as Fuchs'endothelial dystrophy, that require removal of Descemet's membrane in addition to endothelial cells. We will investigate our hypothesis by culturing HCECs in monolayer and transplanting them to human corneas in a perfusion organ culture system of human anterior segments ex vivo. The data obtained from these studies will become the foundation for future endothelial cell transplantation studies in animals, and ultimately in humans. Three specific aims will be investigated in this proposal to test our hypothesis: " Determine the effect of the type, size, and concentration of SPMs on the viability, magnetic attraction, and light transmittance of cultured HCECs in vitro. " Determine the anatomical and physiological outcomes of transferring suspended cultured HCECs with incorporated SPMs to the human perfusion organ culture model of anterior segments. " Determine the mechanism of HCEC attachment to corneal stroma. PUBLIC HEALTH RELEVANCE In this proposal, we will optimize a novel technique of transplanting human corneal endothelial cells, which might become a treatment for many diseases that presently require a corneal tissue transplant. The major advantage of endothelial cell transplantation will be to reduce, and possibly eliminate, corneal donor tissue shortages because the transplanted cells can be grown in a laboratory and used to treat several patients instead of only one patient.